Although glutamate is a major excitatory neurotransmitter in the brain, the specific route of its synthesis is unknown. A possible precursor is ornithine, which is known to be converted in vitro to glutamate via the enzyme ornithine aminotransferase (OAT). We previously found that inhibition of this enzyme in the septum in vivo led to the loss of glutamate, the kinetics of which was sensitive to an acute lesion of the glutamatergic afferents. In this study, tracer techniques were used to determine if the conversion of ornithine to glutamate proceeds in vivo and if canaline blocks such conversion. Intraventricular administration of 3H-ornithine led to the labeling of septal glutamate. This labeling was largely blocked by intraseptal canaline, but the conversion of 14C-Alpha-ketoglutarate to glutamate was unaffected. Thus, canaline specifically blocks the formation of glutamate from ornithine. This finding, in connection with the previous lesion studies, supports the role of ornithine as a precursor for the neurotransmitter pool of glutamate.